An emerging theory in the fields of learning, memory and addiction, is that the regulation of chromatin structure through DNA methylation and histone modification mediate long-lasting behavioral changes. A large number of combinations of posttranslational modifications on histones are possible. This cipher is referred to as the histone code. The methylated component of this code is bound by proteins with Chromo, Tudor, WD40, MBT, PHD domains and have been termed "code-reading proteins". Here we focus on proteins that are expressed in the brain, and that contain these methyl-binding modules, in order to identify those proteins that are capable of being recruited to chromatin after epigenetic changes have been introduced on the histone tails. This study will provide the first glimpse at the effectors of the histone code in the brain. We hypothesize that PHD domain- containing proteins form a prominent group of methyl-dependent histone binding proteins in the brain, and that both arginine and lysine methylation regulate these interactions. AIM 1. Use protein microarrays to identify methyl-dependent binding brain proteins. Proteins that are expressed in the brain, and harbor potential methyl-binding domains, have been identified. These methyl- binding modules (PHD, Tudor, MBT and Chromo domains) will be cloned as GST fusion proteins and arrayed onto glass slides. The resulting microarrays will be probed with biotinylated peptides that represent all the known methylation sites on the core histones, to determine the binding specificity of these domains. Six PHD domain-containing proteins are mutated in different mental retardation syndromes. This genetic data, together with the newly discovered methyl-binding properties of PHD domains, strongly implicate proteins carrying these domains as readers of epigenetic marks in the brain. AIM 2. Determine that the dual R2me/K4me3 mark can be generated in vitro and in cells. The histone H3K4 methyl-mark binds the PHD domains of BPTF and ING2. The methylation of a nearby arginine 2 (R2) residue may regulate these interactions. To confirm that the R2me2 and K4me3 co-exist in cells, we will raise methyl-specific antibodies to the combined H3R2me2K4me3 epitope. Using this antibody, we will assess the prevalence of the duel methyl-modification by Western analysis of core histone isolated from PC12 cells. This antibody will also be used to identify the enzymes that can deposit these marks in vitro. AIM 3. Establish that functional relevance of the dual R2me/K4me3 mark. PRMTs that contribute to H3R2 methylation in vitro will be knocked-down (shRNA) in PC12 cells, individually and in combination, to determine which PRMT is the major contributor to this mark in cells. The subcellular localization of GFP-PHD domain proteins will be monitored in loss- and gain-of-function cells. We will also investigate whether the dual H3R2me2K4me3 modification associates with active or inactive chromatin. The understanding of how we learn, and how memories are retained by the brain remains largely unanswered. These mechanisms are clearly not genetic in nature. The learning process may be achieved through the modification of proteins in the brain, and these different modifications may combine to form a code. In this study we plan to investigate how this memory code is read. This basic scientific study will clearly impact the fields of drug addiction, mental retardation and aging diseases of the brain. [unreadable] [unreadable] [unreadable]